Completed Research Grants
Research Institution: Fondazione Centro San Raffaele del Monte Tabor, Italy
Principal Investigator: Dr Davide Gabellini
Project Title: Molecular Genetic Basis of Facio Scapulo Humeral Dystrophy
Project Status: Completed
Summary: Facioscapulohumeral muscular dystrophy (FSHD) is a genetic disorder involving slowly progressive muscle degeneration in which the muscles of the face, shoulder blades and upper arms are among the most severely affected.
In the majority of genetic disorders, the disease is caused by the absence of a protein due to the fact that the information required for the production of that protein has been lost as a result of gene mutation. On the contrary, FSHD results form excessive protein production. FSHD is associated with reduction in the number of copies of DNA on chromosome 4, called D4Z4, that is repeated may times toward the end of the long arm of chromosome 4 in position 4q35. D4Z4 units are arranged like the carriages of a train. In healthy subjects, there are many D4Z4 units, the “HEALTHY train” goes slowly and protein production from the FSHD region is kept under control. In FSHD patients, there are too few D4Z4 units, the “FSHD train” goes too fast and protein production from the FSHD region goes out of control. In particular, excessive production of two proteins located in the FSHD region (called DUX4 and FRG1) seems to play a role in FSHD onset and progression. Interestingly, various animal models supporting an important role for FRG1 in FSHD have been generated. Indeed, increased production of FRG1 in mice, frogs and worms recapitulates the most prominent features of FSHD (Nature 2006 439:973-7; Dev Dyn. 2009 2381502-12; Dis Model Mech 2009 2:267-74; J Cell Sci 2010 123:1116-23).
Dr Davide Gabellini’s team has been interested in understanding what causes the switch toward excessive DUX4 and FRG1 protein production in FSHD. Indeed, they firmly believe that if they understand how protein production from the FSHD region is regulated they could find ways to bring it back to normal levels in FSHD patients.
Specific Aim 1. Analysis of chromatin modifications at 4q35.
The number of D4Z4 repeats is a critical determinant of the age of onset and clinical severity of FSHD. In general, fewer repeats are associated with a more severe phenotype that presents in childhood. The team has hypothesized that D4Z4 could have different activities according to the number of repeats. To test this hypothesis, they have investigated which proteins are bound to D4Z4 when there are a lot or few repeats. Their results indicate that D4Z4 function by regulating the level of compaction of the FSHD region. In particular, the team has found that D4Z4 is associated to different groups of protein in healthy subjects and in FSHD patients. They are the Polycomb Group (PcG) and Trithorax Group (TrxG) proteins. These are very important proteins that play a fundamental role in controlling protein production during development. Usually, PcG proteins act by increasing the level of compaction of the region where they bind resulting in reduced protein production. Accordingly, the team has found that PcG proteins are bound to D4Z4 in healthy subjects, the region is very compact and protein production from the FSHD locus is kept at very low levels. On the contrary, TrxG proteins act by opening the structure of the region where they bind increasing protein production. Interestingly, they have found that TrxG proteins are bound to D4Z4 in FSHD patients, the region is much more open and as a result protein production from the FSHD locus is increased.
All together, the team’s data indicate that D4Z4 is a platform on which different proteins land according to the number of D4Z4 repeats. When there are many repeats, there is room for landing of PcG proteins, the region is compact and there is low protein production from the FSHD locus. When there are few D4Z4 repeats, there is room for landing TrxG proteins, the region is opened up and there is increased protein production from FSHD region. In particular, there is increased production of DUX4 and FRG1 that cause muscle degeneration and FSHD.
Specific Aim 2. Determination of the biological relevance of the DBE transcript.
This aim focuses on understanding how D4Z4 function in regulating protein production from the FSHD region. The team has found that reduction of D4Z4 units in FSHD is associated to the production of a particular molecule called non-protein coding RNA (ncRNA). They have found the level of the ncRNA are very low in healthy subjects, while there is a great increase in ncRNA production in FSHD patients. Interestingly, they have found that the ncRNA is produced specifically in the muscles of FSHD patients explaining the fact that FSHD is primarily a disease affecting the skeletal muscle. Their data strongly indicates that the ncRNA is responsible for recruiting TrxG proteins to the FSHD locus in FSHD patients and for the opening up of the region. Going back to the analogy with the train, one can think at this ncRNA like at the charcoal that fires the engine. Hence, the team thinks that they have found the reason why in healthy subjects the healthy D4Z4 train goes slowly and there is low protein production from the FSHD region. The reason is that there is very low charcoal (ncRNA) available. Instead, in FSHD patients there is plenty of charcoal (ncRNA), the FSHD D4Z4 train goes faster and there is increased protein production for the FSHD region. Importantly, they have developed ways to specifically reduce the amount of the ncRNA. They have found that if they reduce the amount of the ncRNA, they reduce the speed of the train bringing protein production from the FSHD region close to normal levels. Based on this, they hope that the continuation of their studies will allow them to develop therapeutic approaches for FSHD.
Research Institution: Faculte de Medecine de la Timone, France
Principal Investigator: Dr Frederique Madginier
Project Title: Deciphering the Long-distance Interactions of the D4Z4 Array in control and FSHD Cells
Project Status: Completed
Summary: Facio-Scapulo-Humeral Dystrophy (FSHD) is the third most common myopathy with an autosomal dominant mode of inheritance. FSHD is caused by contraction of an array of repeated sequences, D4Z4, in the terminal region of chromosome 4 (4q35 locus). Several hypotheses have been proposed to explain FSHD. However, the mechanism of this pathology remains elusive and controversial due to the complexity of the 4q35 region and the absence of a clear “FSHD gene”. The lack of candidate genes and the deletion of repetitive elements linked to the disease indicate that FSHD is not the result of a classical mutation within a coding sequence but rather related to changes in chromatin organization and epigenetic alterations, possibly involved in the regulation of the DUX4 gene present within the repeat.
Thanks to FSHD funding, our goal was to test the function of D4Z4 on genome organization and regulation, to characterize the role of the distal variants (4qA and 4qB sequences) and identify new sequences, which might modify or contribute to FSHD pathogenesis and uncover the tri-dimensional organization of the FSHD locus in healthy and diseased muscles.
We recently showed that the 4q35 telomere replicated late during S-phage and that the interaction between D4Z4 and the nuclear lamina directly affects replication timing (Arnoult et al., In revision). We also showed that the 4qA region, which is important for FSHD together with shortening of the D4Z4 array, induces chromatin condensation in a histone-dependent manner but independently of DNA methylation. On the other hand, the 4qB-specific region maintains a more open chromatin structure.
Our objective was then to understand how the reduction of D4Z4 number to a certain threshold reorganizes the chromatin of this locus and leads to the disease. To address these issues we developed the chromatin configuration capture technique (3C) in order to test the interaction of D4Z4 with other 4q35 sequences and identify new sites involved in the disease throughout the genome. These different sites identified by 3C will be confirmed by FISH and analyzed in samples from patients. We hope that this project will help to understand the function of D4Z4 in organizing the chromatin architecture in normal cells and understand how the reduction of this array to a certain threshold of repeats leads to FSHD through the identification of new candidate sequences.
Research Institution: University of Massachusetts Medical School,Massachusetts, USA
Principal Investigator: Professor Rossella Tupler
Project Title: Defining the Mechanism Controlling Muscle-Specific Gene Expression in FSHD
Project Status: Completed
Summary: Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary disease of the muscle. Notably, not all muscles are affected and in a family not all the people that should carry the same genetic defect display muscle weakness.
FSHD has been associated with reduction of the number of sequences composing a tiling array at the end of chromosome 4 long arm.
In the past we found that certain proteins, named YY1, HMGB2 and nucleolin, form a complex that binds the sequence array at chromosome 4 end. We also discovered that genes close to the sequence array become more active when these proteins are removed. We therefore thought that this abnormal activation drive FSHD onset. Consistent with this hypothesis the overactivation of one of these genes, FRG1, can cause muscular dystrophy in the mouse.
Based on these results we considered that discovering factors that can influence the activity of genes like FRG1, might help in finding possible treatments.
We studied the way repeat DNA array is wrapped around special protein, named histones, and treated cells with different drugs. Interestingly we discovered that in cells treated with drugs that damage DNA, genes that are close to the repeat can be activated. We also discovered that this activation is stronger in cells with a reduced number of repeats as FSHD cells.
We also discovered that YY1, the protein, which binds the sequence array at the chromosome 4 end, can also bind a protein, MeCP2, which has been involved in another human disorder which is also due to abnormal activity of genes.
These findings highlight the possibility that through these studies we can find indications on how to prevent or reduce the effect of abnormal gene expression in FSHD patients. This possibility is supported by the observation that the genetic defect, which is present in FSHD patients, not always leads to the development of muscular dystrophy.
Our research will continue to define proteins that are involved in mechanisms that regulate the organization and the activity of genes at the end of chromosome 4 long arm.
Research Institution: Tulane Medical School, New Orleans, USA
Principal Investigator: Professor Melanie Ehrlich
Project Title: Comparing the DNaseI-Hypersensitive Chromatin Landscape at 4q35 of FSHD and Control Cells
Project Status: Completed
Summary: Of the 23 different human chromosomes in each cell, we know that chromosome 4 is the most important for the genetic disease called facioscapulohumeral muscular dystrophy (FSHD). Nonetheless, there is still no consensus among scientists in the field of FSHD about which gene in chromosome 4 is abnormally expressed so as to initiate the disease.
FSHD patients have an abnormally short, disease-causing array of tandem DNA repeats (D4Z4) at one end of this chromosome. We and others consider it very likely that the FSHD-causing gene is within the 4 million base-pairs of this end of chromosome 4, a region that is about 2% of the length of this chromosome. The FSHD gene is also likely to be located at least 80,000 base-pairs from a short, disease-linked D4Z4 array. Complicating matters, there are very few known genes in the entire 4 million base-pair region around D4Z4. Conventional methods to look for an FSHD gene in this region, including in D4Z4 itself, have yielded only controversial conclusions. Therefore, we used new state-of-the-art methods to discover genes and gene-regulatory DNA sequences that might be involved in the disease by identifying special chromosomal sites called DNaseI-hypersensitive sites.
These sites are atypical because they have highly exposed DNA that can get cleaved by the enzyme DNaseI due to the lack of shielding by the usual chromosomal proteins. We tracked these sites on microchips (DNase-chip) and by massive, rapid DNA sequencing of all the human chromosomes (DNase-seq). Application of these techniques to biopsy-derived muscle cells from patients and unaffected individuals revealed a new candidate DNA subregion for FSHD that may interact with short D4Z4 arrays at chromosome 4 to cause the disease. Planned experiments involving this special DNA site and other DNaseI-hypersensitive sites may lead to the identification of the FSHD gene itself and to downstream genes involved in the disease. In addition, we discovered many new regulatory sequences on other chromosomes that are likely to be involved in the repair of muscle in unaffected individuals and in patients.
We also identified some poorly understood genes whose role in muscle formation was not previously known. This first in-depth analysis of the chromosome structure of all the chromosomes in muscle cells should provide new targets for treating FSHD, a painful and crippling disease for which there is currently no effective treatment, and for understanding normal and abnormal muscle formation at the gene level.
Research Institution: Radboud University, The Netherlands
Principal Investigator: Professor Baziel GM van Engelen
Project Title: Biomarkers in FSHD, a Metabolome Study in Blood, Urine and Muscle
Project Status: Completed
Summary: In a collaboration between the departments of Neurology, Psychology, Radiology and Rehabilitation Medicine of the Radboud University Nijmegen Medical Centre, The Netherlands 50 FSHD patients have been enrolled in a randomized controlled trial comparing the effect of home-based cycle ergometer training, cognitive behavioural therapy, and usual care. Blood and multiple urine samples of each of the 50 patients have been sampled for metabolomic analysis to find biomarkers for disease progression and at a better understanding of the pathobiology underlying FSHD. In addition, 30 patients were clinically well characterized and had MR imaging (MRI) as well as MR spectroscopy (MRS) of the upper leg muscles before (n=13), or before and after (n=17) training, cognitive therapy, or waiting list. MR shows the highly variable nature of FSHD between patients, between muscles in patients, and also within muscles.
Main findings are: muscles are binary affected (normal or fatty infiltrated with only a few muscles that were intermediately affected), in intermediate affected muscles fatty infiltration increases linearly in the distal direction (towards the knee), disease progression is fastest in intermediate affected muscles (significant changes occur already within 4 months), fatty infiltration inversely correlates with muscle strength, and PCr/ATP decreases as function of fatty infiltration.
In conclusion, T2 MR muscle imaging seems to be a promising biomarker in FSHD.
Research Institution: Sydney IVF
Location: Sydney, Australia
Principal Investigator: Dr Tomas Stojanov
Primary Focus: Derivation of FSHD-specific human embryonic stem cell (hESC) lines
Status: Currently underway
Summary: Disease specific hES cells are sought to be a powerful tool for research into specific disease mechanisms and as in vitro model systems for drug development in a number of pharmaceutically underserved diseases. The current Sydney IVF project involves the derivation of up to 6 hESC lines from embryos identified by Preimplantation Genetic Diagnosis (PGD) containing the chromosomal rearrangements associated with the FSHD condition. Derivations will be performed under standardised and fully defined culture conditions. Successfully derived stem cell lines will be fully characterised, cryo-banked and expanded to supply the demand for FSHD research worldwide using high content and high throughput systems. Genetic and developmental modifications of these lines by the Sydney IVF lab will further tailor the FSHD-specific cell lines to future applications in drug development and testing assays.
Only eggs that are unusable for IVF because they carry the FSHD mutation will be used. In addition, in all instances donors would have to provide their consent for use of the otherwise discarded eggs.